1. Field of the Invention
This invention is directed to novel immunoconjugates that are useful for diagnosis and therapy. In particular, this invention is directed to immunoconjugates comprising an antibody fragment which is covalently bound to a diagnostic or therapeutic principle through a carbohydrate moiety in the light chain variable region of the antibody fragment. This invention is also directed to immunoconjugates comprising an antibody moiety that is an intact antibody containing a glycosylation site in the light chain variable domain which has been introduced into the antibody by mutating the nucleotide sequence encoding the light chain. This invention is further directed to methods for preparing such immunoconjugates. This invention also is directed to methods of diagnosis and therapy using such immunoconjugates.
2. Related Art
Monoclonal antibodies can be conjugated to a variety of agents to form immunoconjugates for use in diagnosis and therapy. These agents include chelates, which allow the immunoconjugate to form a stable bond with radioisotopes, and cytotoxic agents such as toxins and chemotherapy drugs. For example, cytotoxic agents that normally would be too toxic to patients when administered in a systemic fashion can be coupled to anti-cancer antibodies in such a manner that their toxic effects become directed only to the tumor cells bearing the target antigens. The diagnostic or therapeutic efficacy of immunoconjugates depends upon several factors. Among these factors, the molar ratio of the diagnostic or therapeutic principle to antibody and the antibody binding activity of the immunoconjugate are of major concern.
Researchers have found that the maximum number of diagnostic or therapeutic principles that can be directly linked to an antibody is limited by the number of modifiable sites on the antibody molecule and the loss of immunoreactivity of the antibody. For example, Kulkarni et al., Cancer Research 41:2700-2706 (1981), have reported that there is a limit to the number of drug molecules that can be incorporated into an antibody without significantly decreasing antigen-binding activity. Kulkarni et al., found that the highest incorporation obtained for methotrexate was about ten methotrexate molecules per-molecule of antibody, and that attempts to increase the drug-antibody molar ratio over about ten decreased the yield of immunoconjugate and damaged antibody activity. Kanellos et al., JNCI 75:319-329 (1985), have reported similar results.
In order to achieve a high substitution level of drug-immunoconjugate without significantly impairing antigen-binding activity, researchers have investigated the use of a water-soluble polymeric molecule as an intermediary for the indirect conjugation of the drug. Such polymers include oxidized dextran (Arnon et al., Immunol. Rev. 62:5-27 (1982)), poly-glutamic acid (Greenfield et al., Antibody Immunoconjugates and Radiopharmaceuticals 2:201-216 (1989)), human serum albumin (Baldwin et al., NCI Monographs 3:95-99 (1987)), and carboxymethyldextran (Schechter et al., Cancer Immunol. Immunother. 25:225-230 (1987)).
Shih et al., Int. J. Cancer 41:832-839 (1988), have described a site-specific linking method in which methotrexate was linked to the carbohydrate moiety in the constant, or "Fc," region of an antibody via amino-dextran, resulting in an immunoconjugate with high substitution ratio and retention of immunoreactivity. More recently, Shih et al., Int. J. Cancer 46:1101-1106 (1990), demonstrated the efficacy of an immunoconjugate comprising 5-fluorouridine conjugated via amino-dextran to the carbohydrate moiety in the Fc region of a monoclonal antibody. In both studies, Shih et al. found that the immunoconjugate contained approximately 30-50 molecules of drug per molecule of immunoglobulin. Thus, indirect conjugation of a diagnostic or therapeutic principle to a carbohydrate moiety in the Fc region of an antibody provides a means to obtain immunoconjugates with functional antigen binding activity and a high substitution level.
An advantage of using the carbohydrate moiety in the Fc region as a site-specific attachment site is that antibodies of all subtypes typically contain a glycosylated Fc region. In general, antibody molecules are glycosylated in their Fc regions at characteristic positions according to their isotype. For example, carbohydrate is typically present at amino acid 297 in the C.sub.H 2 domain in the Fc region of IgG molecules. Conjugating a diagnostic or therapeutic principle to the carbohydrate group at this position, which is far away from the antigen binding site, should produce a minimal effect on the immunoreactivity of the resultant immunoconjugate, as demonstrated by Shih et al.
However, a disadvantage of using the carbohydrate moiety in the Fc region as an attachment site is that the entire antibody is required for the immunoconjugate. The use of antibody fragments, particularly Fab, Fab' and F(ab').sub.2 provide an advantage over the use of an entire antibody because such fragments are better able to diffuse out of capillaries and into target tissues. For example, see Brown, "Clinical Use of Monoclonal Antibodies," in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al., eds. Chapman and Hall, pp.227-249 (1993). Moreover, antibody fragments will clear from blood and normal tissues more readily than an entire antibody. For example, intact murine IgG has a blood half-life of approximately 30 hours, while F(ab').sub.2 and Fab/Fab' have half-lives of approximately 20 hours and 2 hours, respectively. Id. Thus, it is advantageous to use antibody fragments for constructing immunoconjugates. Antibody fragments are particularly advantageous in radioimmunotherapy and radioimmunodiagnosis applications in which the exposure of normal tissues to radioisotopes must be minimized.
Antibody variable regions occasionally contain carbohydrate groups which provide potential attachment sites for the preparation of immunoconjugates from antibody fragments. For example, asparagine-linked carbohydrate acceptor sequences have been identified in approximately 15-25% of murine variable regions. Kabat et al. SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th ed. U.S. Department of Health and Human Services (1990). In the case of the anti-dextran family of antibodies, glycosylation sites reside in the complementarity-determining regions (CDRs), particularly CDR2, of the heavy chain variable regions. Id. The presence of Asn-linked carbohydrates in the CDRs of these antibodies appeared to enhance antigen binding. Wallick et al., J. Exp. Med. 168:1099-1109 (1988); Wright et al., EMBO J. 10:2717-2723 (1991). However, introduction of additional carbohydrate attachment sites in CDR2 by site-directed mutagenesis resulted in either the enhancement or reduction of affinity for antigen, depending on the position where the glycosylation site was introduced. Wright et al., supra. Thus, the feasibility of attaching a diagnostic or therapeutic principle to a carbohydrate moiety in the VH CDR region is uncertain.
Studies by the present inventors on carbohydrate conjugation demonstrated a high conjugation efficiency with the IgG antibody, LL2, which is a murine monoclonal antibody described by Pawlak-Byczkowska et al. (Cancer Res. 49:4568-4577 (1989)) and Goldenberg et al. (J. Clin. Oncol. 9:548 (1991)). Analysis of LL2 conjugates using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions indicated the existence of a glycosylation site in the light chain variable (VL) region of the LL2 antibody. After cloning the VL region of LL2, an Asn-linked glycosylation site at position 18-20 of the framework-1 (FR1) sequence of the VL region was found.
These studies suggested a possible preferential conjugation at a carbohydrate moiety within the VL region. This unexpected finding may be explained by an improved accessibility in the VL region. We used site-directed mutagenesis to remove the Asn-linked glycosylation site and found that the resulting protein exhibited similar immunoreactivity compared with the original antibody. This result is in agreement with the inventors' computer modeling studies which suggested negligible or minimal interaction between the light chain FR1 carbohydrate moiety and the antigen binding site. Thus, these studies indicate that conjugation of a diagnostic or therapeutic principle to a carbohydrate moiety in the FR1 sequence of the VL region provides a means to obtain immunoconjugates of antibody fragments with functional antigen binding activity.
The present invention provides a method for preparing novel immunoconjugates comprising a diagnostic or therapeutic principle which is attached to an intact antibody, or antigen-binding fragment thereof, via a carbohydrate moiety of the light chain variable region.